Freezable live cell diluent and process



United States Patent Qffice 3,444,039 Patented May 13, 1969 3,444,039FREEZABLE LIVE CELL DILUENT AND PROCESS Ambrose Harry JesudasanRajamannan, Anoka, Minn. (103 Landmark Drive, Eastern Heights, Ithaca,N.Y. 14850) No Drawing. Filed Dec. 14, 1964, Ser. No. 418,304 Int. Cl.C12k 9/00; A01n 1/02 U.S. Cl. 1951.8 18 Claims ABSTRACT OF THEDISCLOSURE This invention relates to new and useful improvements in theart and method of preservation and freezing of live mammalian cellmaterials with partially hydrolyzed starches and essentiallypolysaccharides, and the products obtained thereby. More particularly,this new disclosure to the art concerns the provision of improved andmore economical extender and preservative diluent material as freezablenon-growth live cell preservative diluent, compounded in the criticalrelationship of an acidic solution of a mixture of predominantlycarbohydrate polysaccharides and a buffering agent conditioning themixture to a critical pH range of 6 to 7.5, and which contains over 80%carbohydrate polysaccharides exclusive of mono and disacch'arides andinclusive of at least 30% mixture of Tri to Hepta saccharides, saidmixture in solution, having an osmotic pressure in the critical range of225 to 350 milliosmoles; including the combination therewith of a liquidextract from dry solids of egg yolk for mixing with such live cell andcell tissue matter as semen, germ plasma, bone marrow cells, muscle andartery tissue cells, and the like cell matters, which live cell mattersare killed or damaged beyond practical use in the presence of thepartially hydrolyzed vegetable starches constituting the high quantitypolysaccharides when utilized without such conditioning, the method ofpreparing the said critically buffered preservative diluent material,and the combination therewith of such live cell materials.

The art has particularly been faced with the problem of cell damage inpreservation and freezing in sugar, alcohol and other preservativesheretofore disclosed and known to the art. One of such prior disclosuresis that found in Canadian Patent No. 682,906 issued Mar. 24, 1964. Insuch disclosure the problem of metabolism has led to the use ofexpensive and costly mediums which include primarily polyhydric alcoholsof 4 to 7 carbon atoms, as sorbitol, mannitol, inositol, erythritol andthe like. The prior art also includes the use of such materials asglucose, fructose, adonitol, and the like. In general, the fault withsuch materials is not only the cost factor, as there has remained themajor problem of a decrease in healthy cells recovered after freezing.Even in the most expensive preservatives perfected, a high percentage ofthe cells recovered after freezing are injured and defective. It willthus be recognized that an alternative and new preservative diluentformulation of greater economy in manufacture and at the same timeproviding an improved recovery of live healthy cells would be of newadvantage and is of need in the art.

Accordingly, it is the principle purpose of this disclosure to providethe art with improved preservative diluents which are economical toproduce and by their use provides an improvement in the recovery ofhealthy l-ive mammalian cells, including live tissue cell matter, afterpreservation by freezing in admixture with the diluents, as hereinprovided.

Another object of this disclosure is to provide a method of preparingthe preservative dilnents herein provided from a low cost mixture ofnaturally acidic carbohydrate polysaccharide material, of predominantlypolysaccharides obtained by partial hydrolysis of naturally occurringvegetable starches and obtaining herewith an improved economicalpreservative diluenlt for live mammalian cells and which affords animprovement in recovery of healthy live sells after freezing.

In general, it may be stated that to obtain the new and useful objectsand advantages of the method and preservative diluents herein providedcomprises the preparation of a very mildly alkaline or buffered solutionof an essentially normally acidic mixture of said carbohydratepolysaccharides material, as a mixture of partially converted hydrolysisproducts of naturally occurring starches of the vegetable kingdom, inthe critical pH range of 6 to 7.5 and with an osmotic pressure betweenthe critical range of 225 to 350 milliosmoles, in combination with anextract of a liquid fraction from spray dried egg yolk solids, andprepared in a temperature range of 2 C. to 37 C. This diluent andpreservative is thereafter combined and used with the indicated livecell materials in a conventional manner. The preferred use of theprepared diluent, with added live cell matter, is in the freshlyprepared freezable liquid form. However, the liquid portion of theprepared diluent may be evaporated, as by spray drying, or freezedrying, and the diluent solids later dissolved in water ready forextender and preservative use.

For the purpose of exemplificati-on, the said mixtures of carbohydratepolysaccharides composition, in prepared form as preservative diluents,will be hereinafter illustrated by their advantageous use in the fieldof pre serving animal bovine semen for insemination.

To the accomplishment of the foregoing and related ends, thisimprovement in the art therefore comprises the features hereinafter morefully described and particularly pointed out in the claims, thefollowing description setting forth in detail certain illustrativeembodiments provided in this disclosure, these being indicative,however, of the various ways in which the principles of this disclosuremay be employed.

To illustrate by specific examples the following are provided:

EXAMPLE 1 Sodium citrate grams 54 Water cc. 2,000 Spray dried egg yolk"grams" 100 Partially hydrolized starch solids (grams per 1,000

cc. liquid extract) 5 The basic buffer solution of sodium citrate andwater was prepared at slightly below normal room temperature. Then, theegg yolk material was dispersed in the prepared buffer solution. Uponmixing, a coagulum forms which is separable from the clear liquid bysuch conventional means as centrifuging, filtering or decanting. In thisinstance the mixture was allowed to stand until it had separated intotwo parts, (1) an upper coagulum fraction and (2) a lower clear liquidfraction. The lower clear fraction was extracted and to each 1,000 cc.clear fraction of extract was added 5 grams of partially hydrolyzedstarch in the form of corn syrup solids and which are essentially overpolysaccharides. This constitutes a liquid diluent which is ready foruse in a conventional manner, or in preparation for a live cell freezingprocess. If it is desired to freeze the solution of preservative diluentand live cell matter mixed therein, there is added about 5% to glycerolby volume. The glycerol may be added before or after the mixture of livecell matter with the prepared liquid preservative diluent.

EXAMPLE 2 Alkaline citrate salt "grams" 27 Water cc 1,000 Egg yolksolids grams 50 Corn syrup solids do 5 The above components may bemerely added together and mixed. However, it is preferred to prepare thebase buffering solution by mixing the alkaline citrate salt, as sodiumor potassium citrate in the Water and then add corn syrup solids andspray dried egg yolk solids followed by thorough mixing. After mixing,the mixture is allowed to stand until it has gradually separated intotwo fractions, as above indicated. The lower clear fraction is extractedand ready for use, with the desired amount of live cells added. Theaddition of the cell matter to an extender liquid, in any amount, forpractical use is optional and known to the art. If it is desired tofreeze the mixture of preservative diluent and cells, about 5% to 10%glycerol, by volume, is added.

The following illustration will show how some modification of the solidsportion may be made:

EXAMPLE 3 Buifering agent to pH 6 to 7.5 and osmotic pressuremiliiosmoles 250-300 Water cc 1,000 Egg yolk solids ms 25-100Carbohydrate polysaccharides (defined) (preferably 2 to grns. if nocomplementary sugar or alcohol is added) gms .5-15 Fructose and/ orglucose grns 02 The above components were mixed as in Example 2including separation of the desired liquid extract. The extract may alsobe prepared in the manner of Example 1. The buffering agent, as utilizedherein, may be any suitable salt or alkaline agent, or mixtures of thesame. For example, such agents as citrates, acetates, phosphates,carbonates, and the like may be used to neutralize the otherwise acidsperm killing nature of the saccharides and effect the pH control, asdescribed. The carbohydrate saccharides are a mixture of a minimum ofmono and disaccharides with a maximum of tri, tetra, penta, hexa, heptaand higher polysaccharides. The products utilized herein are obtained bypartial acid and/ or partial enzymic hydrolysis of naturally occurringstarches as obtained from corn, maize, potatoes, wheat, rice, tapioca,oats, and other plant and vegetable matter of the vegetable kingdom. Thehydrolysis products are a partial conversion between 10% to 50% dextroseequivalent. This mixture The process of Example 1 using sorghum syrup inthe formula proportion of Example 2.

In the above formulations a faster wetting of mixtures with the driedegg yolk solids was noticed when the said partially hydrolyzed starchmaterials were added, in the stage of effecting extraction of the driedegg yolk solids. This did appear to measurably reduce the mixture time.In view of the relative similarity in production of partial hydrolysisof the vegetable starches, it is not believed to be necessary to furtherdescribe other formula and similar naturally occurring starches in theform of use as partially hydrolyzed starches and polysaccharides asherein described.

In the illustrative specific examples the relative percentages of solidsare on the order of about 33% buffering agent, about dried egg solidsand about 6% saccharides solids. In general, the relative solids mayvary on the order of 87% to 96% egg yolk solids to about 3.8% to about13% carbohydrate polysaccharides, with the buffering agent present in anamount sufficient to effect a pH in the critical range value of 6 to 7.5and the solution in water having an osmotic pressure in the criticalrange of 225 to 350 milliosmoles.

The preparation of preservative and extender mediums in combination withlive cell materials is Well known to the art and as described in theabove mentioned patent. The particulars therefore, in preparing livecell mixtures of the herein described preservative diluent and extenderare optional with regard to concentrations of cell matter and diluent,as is known to the particular field of use in preservation and extendingof live mammalian cell matter. For the above tests the dilution was 1part semen to 100 parts preservative and extender diluent, as iscommonly used for insemination of bull semen. The application forinsemination is otherwise of the same order as known to the art.

In tests of bovine semene in admixture with the herein describedpreservative diluent material, compared to like semen tests of astandard yolk citrate medium and another called Minnesota Go, a popularsugar alcohol extender, the following results were obtained:

1. With Low Quality Semen Applicant's (Bovine) 1. Std. Yolk Citrate 2.Minn. Go" 3. Improved Diiusnt of Example 1 St a e Ambi tT 1 dayViability 2 day Viability 2 day Viability. Plifs 5 0 2 3 day Viability 5day Viability 7 day Viability.

2. High Quality Semen (Bovine) Storage Ambient Temp 1 to 2 days 3 days 3days. Plus 5 C 5 days 7 days 7 days.

POST FREEZING SURVIVAL 3. High Quality Semen (Bovine) Before Freezing607 Motility 60% Motility 60% Motility. Recovery After Freezing toLiquid 309?; Motility 40% Motility 55%Mot1l1ty.

Nitrogen Temp.

4. Low Quality Semen (Bovine) Before Freezing 40% Motility 40% Motility40% Motility. Recovery After Freezing 5% Motility 20% Motility 35%Motility.

Progressive type of motility showing no sperm cell damage and an amazingincrease in survival capacity.

The proof of the advantages which are gained by the herein described newand useful preservative diluent is illustrated from the post freezingsurvival pattern in low quality semen. Bulls of high performance(normally associated with low quality semen) can be utilized to a morebeneficial extent since the freeze kill and cell damage is minimized.That is, the freeze kill has been reduced and a higher percentage oflive cells are recovered, enabling a high quality semen producing bullto be used on a larger scale. Under the frozen semen program, the lowquality semen producing bulls, many valued at $10,000 or more, wouldotherwise be discarded, with heretofore known and utilized semenpreservative medium, despite high pedigree and performance. However, byuse of my new preservative diluent compositions, such bulls can be savedand their semen used more productively.

In similar experimental tests with like diluted mixtures of frozen livecell matter, as the semen of cocks, studs, rabbits, pigs, dogs, rams andgoats, with the carbohydrate saccharide medium herein provided, therecovered unfrozen mixtures, under microscope analysis, showed acomparable progressive type of motility with high activity and noappreciable cell damage.

Illustrative of a different application, a small sample of blood wastaken and mixed with the prepared carbohydrate polysaccharidecomposition, minus the egg yolk extract, in the proportion of about 1part blood to 2 parts of said composition. This mixture was frozen underliquid nitrogen temperature condition. Upon microscopic comparison ofthe unfrozen blood cells, with a new fresh sample from the same bloodsource, no visible cell damage was apparent. Inasmuch as thecarbohydrate polysaccharide material may be a normal human foodmaterial, in its liquid state it may also be used as a preservative inthe field of frozen blood for transfusion and frozen preservation ofmuscle, ligament and tissue cell matter, under suitable hygienicconditions and with or without a suitable antibiotic, as known to theart. T 0 those skilled in the art, the equivalent and similarity of thepreservative diluent for use with mammalian cell material will berecognized.

From the above description and subject matter of disclosure it will beapparent that some modifications as herein set forth may be made withoutdeparting from the spirit and scope thereof, as embodied in the terms ofthe following claims.

I claim:

1. In the process of preparing a freezable diluent for live cells thesteps of preparing a water solution with an alkaline buffering agent,mixing dried egg yolk in the solution, effecting a separation of asolids portion and a clear liquid portion of said mixture, removing theclear liquid portion from said solids portion, retaining the clearliquid fraction and adding thereto the partially hydrolyzed products ofnaturally occurring starches consisting principally of over 80%polysaccharides of which over 30% consist of tri, tetra, penta, hexa andhepta saccharides.

2. The method of preparing a preservative diluent for live mammaliancells comprising preparing a water solution of dried egg yolk solids anda buffering agent, extracting the liquid from the solids, and addingfrom about 0.5 part to parts water soluble carbohydrate polysaccharidematerial per about each 1000 cc. of water, the addition of saidbuffering agent in relationship to said polysaccharide material beingadded in an amount to effeet a pH value of said solution in the range of6 to 7.5 and an osmotic pressure of 225 to 350 milliosmoles.

3. The method of claim 2 wherein the proportion of said polysaccharidematerial is added between the range of 0.5 to 15 parts with acomplementary sugar.

4. The method of preparing a freezable preservative diluent for livecells comprising preparing a water solution of a buffering material,dried egg yolk solids and carbohydrate polysaccharide material,separating the solids from the liquid and retaining the separated liquidas the preservative medium having a pH value in the order of 6 to 7.5and an osmotic pressure of 225 to 350 milliosmoles.

5. The product of claim 4 including live cells contained in a frozenstate with said medium.

6. A freezable preservative and extender diluent for live cellscomprising a mixture of a combination of water soluble carbohydratepolysaccharides and a buffering agent therefor, said mixture in a watersolution having a pH value of 6 to 7.5 and an osmotic pressure of 225 to350 milliosmoles.

7. The product of claim 6 containing a proteinaceous egg yolks.

8. The product of claim 6 including in combination therewith livemammalian cells in a frozen state.

9. A freezable preservative and extender diluent for live bovine spermcells comprising a mixture of a water soluble extract from dried eggyolks, water soluble carbohydrate polysaccharide material, an alkalinebuffering agent for said polysaccharide material, said mixture in awater solution having a pH value in the range of 6 to 7.5 and an osmoticpressure in the range of 225 to 350 milliosmoles.

10. The product of claim 9 wherein the said carbohydrate polysaccharidematerial is selected from the group consisting of the partiallyhydrolyzed end products of natural starches of the vegetable kingdom.

11. The product of claim 9 wherein the carbohydrate polysaccharidesmaterial is corn syrup material.

12. The product of claim 9 wherein the carbohydrate polysaccharidematerial is the partially hydrolyzed end product of corn starch.

13. The product of claim 12 including live mammalian cells containedwith the said diluent in a frozen state.

14. An improved freezable preservative and extender diluent for livemammalian cells comprising a combination in the proportion of a waterextract of about 25 to parts dried egg yolk solids, about 0.5 to 15parts Water soluble carbohydrate polysaccharide material, and abuifering agent for said polysaccharide material providing a pH value inthe range of 6 to 7.5 in a water solution.

115. The product of claim 14 including live mammalian ce s.

16. The product of claim 14 wherein the carbohydrate polysaccharidematerial is selected from the group consisting of partially hydrolyzednatural starches obtained from the vegetable kingdom.

17. The product of claim 14 wherein the carbohydrate polysaccharidematerial is present in a proportion of about 0.5 to 15 parts incombination with up to about 2 parts complementary sugar.

A freezable preservative diluent for live cells contalmng apolysaccharide material comprising a partially hydrolyzed vegetablestarch with a 10-50% dextrose equivalent and a buffering agent, saidpreservative in a water solution having a pH value of 6 to 7.5 and anosmotic pressure of-225 to 35 0 milliosmoles.

References Cited UNITED STATES PATENTS 3,185,623 5/1965 Smith et al.16753 3,005,756 10/ 1961 Van Demark et al. 16774 FOREIGN PATENTS 368,6726/1961 Japan.

F. CACCIAPAGLIA, JR., Primary Examiner.

US. Cl. X.R. 62-1; 1.7

